The references cited in the present application are not admitted to be prior art to the claimed invention.
It is estimated that about 3% of the world's population are infected with the Hepatitis C virus (HCV). (Wasley et al., 2000. Semin. Liver Dis. 20, 1-16.) Exposure to HCV results in an overt acute disease in a small percentage of cases, while in most instances the virus establishes a chronic infection causing liver inflammation and slowly progresses into liver failure and cirrhosis. (Iwarson, 1994. FEMS Microbiol. Rev. 14, 201-204.) Epidemiological surveys indicate HCV plays an important role in hepatocellular carcinoma pathogenesis. (Kew, 1994. FEMS Microbiol. Rev. 14, 211-220, Alter, 1995. Blood 85, 1681-1695.)
The HCV genome consists of a single strand RNA about 9.5 kb in length, encoding a precursor polyprotein about 3000 amino acids. (Choo et al., 1989. Science 244, 362-364, Choo et al., 1989. Science 244, 359-362, Takamizawa et al., 1991. J. Virol. 65, 1105-1113.) The HCV polyprotein contains the viral proteins in the order: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
Individual viral proteins are produced by proteolysis of the HCV polyprotein. Host cell proteases release the putative structural proteins C, E1, E2, and p7, and create the N-terminus of NS2 at amino acid 810. (Mizushima et al., 1994. J. Virol. 68, 2731-2734, Hijikata et al., 1993. Proc. Natl. Acad. Sci. USA 90, 10773-10777.)
The non-structural proteins NS3, NS4A, NS4B, NS5A and NS5B presumably form the virus replication machinery and are released from the polyprotein. A zinc-dependent 30 protease associated with NS2 and the N-terminus of NS3 is responsible for cleavage between NS2 and NS3. (Grakoui et al., 1993. J. Virol. 67, 1385-1395, Hijikata et al., 1993. Proc. Natl. Acad. Sci. USA 90, 10773-10777.)
A distinct serine protease located in the N-terminal domain of NS3 is responsible for proteolytic cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions. (Barthenschlager et al., 1993. J. Virol. 67, 3835-3844, Grakoui et al., 1993. Proc. Natl. Acad. Sci. USA 90, 10583-10587, Tomei et al, 1993. J Virol. 67, 4017-4026.) RNA stimulated NTPase and helicase activities are located in the C-terminal domain of NS3.
NS4A provides a cofactor for NS3 protease activity. (Failla et al., J. Virol. 1994. 68, 3753-3760, De Francesco et al., U.S. Pat. No. 5,739,002.)
NS5A is a highly phosphorylated protein conferring interferon resistance. (De Francesco et al., 2000. Semin Liver Dis., 20(1), 69-83, Pawlotsky 1999. J. Viral Hepat. Suppl. 1, 47-48.)
NS5B provides an RNA-dependent RNA polymerase. (De Francesco et al., International Publication Number WO 96/37619, published Nov. 28, 1996, Behrens et al., 1996. EMBO 15, 12-22, Lohmann et al., 1998. Virology 249, 108-118.) An important feature of HCV is its high level of genetic variability, which is believed to be a consequence of the low fidelity of the viral polymerase. This variability is underscored by the identification of six major HCV genotypes (designated 1 through 6), more than fifty subtypes, and numerous quasi-species within each subtype. (Farci et al., 2000. Sem. Liver Dis. 20, 103-126.)
Several HCV clones that are infectious in chimpanzees have been described. (Kolykhalov et al., 1997. Science 277, 570-574, Yanagi et al., 1998. Proc. Natl. Acad. Sci. USA 94, 8738-8743, Hong et al., 1999. Virology 256, 36-44.) Isolates based on HCV-con1 and HCV-N have been shown to replicate robustly in cell culture. (Lohmann et al., 1999. Science 285, 110-113, Ikeda et al., 2002. J. Virol. 76, 2997-3006, Guo et al., 2001. J. Virol. 75, 8516-8523.)
Efficient replication in cell culture has been invariably associated with adaptive mutations that dramatically increase the frequency with which replication is established. (Ikeda et al., 2002. J. Virol. 76, 2997-3006, Blight et al., 2000. Science 290, 1972-1974, Lohman et al., 2001. J. Virol. 75, 1437-1449, Kriege et al., 2001. J. Virol. 75, 4614-4624.) Adaptive mutations in the HCV-con1 isolate have been localized to various non-structural genes, though substitutions upstream of the interferon sensitivity determining region in NS5A, for example S232I, appear to be the most effective. (Blight et al., 2000. Science 290, 1972-1974.) Similarly, a 4 amino acid insertion in NS5A that is not commonly observed in vivo is important for replication in cell culture of the HCV-N isolate. (Ikeda et al., 2002. J. Virol. 76, 2997-3006.)